An analysis of intramembrane particles seen in membrane fracture faces was begun this year with the freeze-fracture study of two model membrane systems containing integral, transmembrane proteins cytochrome oxidase and photosynthetic reaction centers. Triton extraction of bovine mitochrondria yields membranous cytochrome oxidase vesicles which often display crystalline arrays of protein. Unusual fracture patterns were observed: Fracture planes in crystalline regions occur at the surfaces of membranes, not within the membrane hydrophobic core, and fracture 100 A steps jump across the entire thickness of the membranes. In non-crystalline regions the fracture planes pass into the hydrophobic core producing large, irregularly shaped particles about 120 A diameter which are composed of four cytochrome oxidase monomers. The irregular shape is probably in part due to plastic deformation and to associated lipid. Bacterial (Rhodopseudomonas spheroides) photosynthetic reaction centers, reconstituted into egg lecithin multilamellar phases, produced oblong intramembrane particles averaging about 60 x 120 A. Comparison with X-ray and neutron diffraction data of oriented multilamellar phases (Dr. J. Pachence, Ph.D. Thesis, Dept. Chemistry, University of Pennsylvania) reveals that 2-3 protein units (each transmembrane about 60 A long and 40 A diameter) aggregate to form a particle. The above samples, as well as peripheral nerve myelin, retinal photoreceptor cells, macrophages, red blood cells, lens junctional complexes and lipid model phases containing alkanes, have been prepared for freeze-fracture in the absence of fixatives or cryoprotectants using ultra-rapid freezing methods. Small samples are sandwiched between copper sheets and rapidly plunged into liquid propane (minus 190 degrees C). Direct measurements using miniature embedded thermocouples indicate that cooling rates are in excess of 10,000 degrees C/s (0 degrees C to minus 100 degrees C). Diffraction and microscopic studies show that damage due to ice crystal growth is avoided in these samples.